The long term of these studies is to understand and target the immunosuppressive activity of leukocytes with phenotypic attributes of inflammatory dendritic cells (DCs) that accumulate at tumor beds. This proposal focuses on elucidating and targeting the mechanisms of inhibition of ?? T cells by members of the butyrophilin family overexpressed in regulatory DCs and macrophages in ovarian cancer. Key findings for the ongoing grant have been: 1) The demonstration that novel antibodies targeting BTN3A1/2/1 rescue ?? T cells from butyrophilin- mediated suppression; 2) the identification of members of the TCR complex and receptor phosphatases that co-IP with BTN3A1; and 3) the finding that BTN3A1/2/3 are universally overexpressed in inflammatory DCs and other myeloid cells in ovarian cancer. Based on these and other findings, our central hypothesis is that BTN3A1 inhibits tumor-reactive T cells by engaging a multimeric CD45 phosphatase:CD3:TCR complex, which antagonizes phosphorylation-dependent TCR signal transduction by increasing the density of CD45 at the TCR network. In contrast, antibody-induced conformational changes in BTN3A1 elicit the activation of V?9V?2 T cells. Accordingly, our Specific Aims are: In Specific Aim 1, we will determine the anti-tumor effectiveness of targeting human BTN3A1 in vivo in tumor-bearing hosts. In Specific Aim 2, we will elucidate the molecular mechanism of T cell inhibition mediated by BTN3A1, based on the identification of a multimeric complex that co-IPs with BTN3A1. In Specific Aim 3, define the mechanism of BTN3A1 deregulation at tumor beds. Our work could exert a profound effect in the field by elucidating a novel targetable immune checkpoint inhibitory pathway mediated by butyrophilins universally overexpressed in ovarian cancer. This mechanistic rationale will pave the way for a novel generation of immunotherapies that could benefit patients currently resistant to established immune checkpoint inhibitors, which may be applicable other lethal tumors and could be relevant for other ?orphan? B7 ligands.